In response to environmental stress such as heat shock, cells must alter gene expression in order to remain viable. A critical control point for regulating gene expression in eukaryotic cells is at the level of mRNA transcription by RNA polymerase II. Most regulators of transcription are proteins. In a break with this paradigm, the heat shock responsive mouse B2 RNA has been found to associate with RNA polymerase II and inhibit transcription after heat shock in mouse cells. My in vitro studies show that B2 RNA inhibits mRNA transcription by binding to RNA polymerase II, incorporating into preinitiation complexes, and blocking transcript synthesis. To understand how small RNA can regulate transcription the studies proposed here will investigate the mechanism by which B2 RNA inhibits transcription by RNA polymerase II in cells after heat shock, map the interaction surface between B2 RNA and RNA polymerase II, and mutate the interacting region on B2 RNA. The specific aims are: 1) to elucidate the molecular mechanism by which B2 RNA inhibits transcription by RNA polymerase II in cells, and 2) to map and mutate the functional interaction surfaces on B2 RNA and RNA polymerase II.